Cardiac myofibrillogenesis is spatiotemporally modulated by the molecular chaperone UNC45B

Figures

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  Figure 1. The stepwise process of cardiac myofibrillogenesis in human embryonic stem cell-derived cardiomyocytes (A–H) Representative immunofluorescence (IF) images of WT-CMs cultured on Matrigel-coated cover slides and harvested at the indicated time points. Cells were stained for (A) UNC45B and KIND2, (B) MYH6 and MYH10, (C) UNC45B and ITGB1, (D) UNC45B and ACTN2, (E) TTN-N and ACTN2, (F) ACTN2 and F-ACTIN, (G) TTN-C and MYH6, and (H) UNC45B and MYH6. Overlapping fluorescence signals in each panel are indicated by arrowheads. All fluorescence images have been merged with DAPI staining. Scale bars: 10 μm. (I–K) Schematics of sarcomere assembly and integrity at (I) day 5 (the premyofibril stage), (J) day 7 (nascent myofibril), and (K) day 10 (mature myofibril). Results represent data from at least three independent experiments. See also Figures S1 and S2.

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  Figure 2. Ablation of UNC45B from hESCs (A and B) Transcription (A) and protein (B) levels of UNC45B in WT hESCs during directed cardiac differentiation from day 1 to day 7, as determined by qRT-PCR (n = 3) and western blot. Error bars represent SD. (C) DNA sequencing data of genomic DNA samples from the two homozygous UNC45B-knockout lines. Black arrows indicate the sgRNA cleavage sites. (D) Protein levels of UNC45B in WT-CMs and UNC45B−/−-CMs, as determined using western blot. (E) Representative IF images of replated WT-CMs and UNC45B−/−-CMs harvested at day 30 and stained for TNNT2 and ACTN2. Scale bars: 10 μm. (F) Representative TEM images of WT-CMs and UNC45B−/−-CMs. Scale bars are indicated. See also Figure S3.

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  Figure 3. ACTN2 accumulation and defective F-ACTIN polymerization during the nascent myofibril stage in UNC45B−/−-CMs (A and B) Representative IF images of MYH6, MYH10, and TTN-C in non-replated WT-CMs and UNC45B−/−-CMs harvested at day 7 or day 10. Cells were stained for (A) MYH6 and MYH10 and (B) TTN-C and MYH6. To improve visualization of MYH6 signal in the UNC45B−/− cells, additional sets of MYH6 images with a longer exposure time are shown. Scale bars: 10 μm. (C) Representative IF images of non-replated WT-CMs and UNC45B−/−-CMs harvested at day 7 or day 10 and stained for TTN-N and ACTN2. Scale bars: 10 μm. (D) Quantification results for ACTN2 signal intensity in WT-CMs and UNC45B−/−-CMs at day 7, showing ACTN2 accumulation (n = 150). Error bars represent SD. Statistical significance is indicated: ∗∗∗p < 0.001. (E and F) Left panel: representative IF images of WT-CMs and UNC45B−/−-CMs harvested at (E) day 7 and (F) day 10 and stained for ACTN2 and F-ACTIN. Scale bars: 10 μm. Results represent data from at least three independent experiments. See also Figure S3.

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  Figure 4. UNC45B-regulated genes mainly involved in heart development, extracellular matrix organization and focal adhesion (A) Scatterplot illustrating the DEGs based on RNA sequencing results from day 7 and 30 WT-CMs and UNC45B−/−-CMs. Up-regulated and down-regulated genes (adjusted p < 0.05) in UNC45B−/−-CMs relative to WT-CMs are shown in orange and blue, respectively. (B) Venn diagram analysis of UNC45B-regulated genes on the basis of day 7 (pink circle) and day 30 (yellow circle) RNA-seq datasets. (C) Gene Ontology (GO) biological process analyses of down-regulated genes in day 7 and day 30 UNC45B−/−-CMs. (D) Hierarchical clustering analysis of the DEGs from day 7 and day 30 RNA-seq datasets. Scale bar represents the Z score value. For RNA-seq, WT-CMs (n = 2), and UNC45B−/−-CMs (n = 2). (E) Ingenuity canonical pathway analysis of DEGs from day 7 and 30 RNA-seq datasets. Gene lists from up-regulated and down-regulated genes (p < 0.05) in UNC45B−/−-CMs relative to WT-CMs. (F) DEG enrichment network analysis of day 7 RNA-seq data reveals that these DEGs are involved in both down-regulated pathways and up-regulated pathways. (G and H) Reactome (G) and KEGG (H) pathway analysis of day 7…

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  Figure 5. UNC45B modulates protocostamere formation by regulating KIND2 expression (A) Co-IP assay showing the association of UNC45B-FLAG with MYH6 and heat shock proteins, as detected using western blots. Cell lysates of WT (negative control) and WTUNC45B-OE-CMs (input) and pull-down samples using anti-FLAG beads (elute) were analyzed using SDS-PAGE. Anti-FLAG antibody was used to validate the co-IP assay. WT-CMs acted as a negative control. (B) Representative IF images of WT-CMs harvested at day 7 and stained with antibodies for UNC45B and HSP90AA1 or HSP70. Scale bars: 10 μm. Enlargement scale bars: 5 μm. Results represent data from at least three independent experiments. (C) Western blot following UNC45B-FLAG and KIND2 pull-down assay. (D) Representative IF images of WT-CMs and UNC45B−/−-CMs harvested at day 7 and stained for KIND2, ITGB1 9EG7, and ACTN2. Nucleation sites are indicated by arrowheads. Scale bars: 10 µm. (E) Representative fluorescence intensity three-dimensional (3D) surface plots of KIND2 and ITGB1 9EG7 signals in day7 WT-CMs and UNC45B−/− CMs, illustrating protocostamere sites. (F) Quantification results of numbers of KIND2 and ITGB1 nucleation sites per CM in WT-CMs…

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  Figure 6. Ectopic expression of full-length KIND2 in a UNC45B−/− line rescues the protocostamere phenotype (A) Western blots of day 7 UNC45B−/−-CMs and UNC45B−/−:KIND2 OE-CMs. (B) Representative IF images of non-replated WT, UNC45B−/−-CMs, and UNC45B−/−:KIND2 OE-CMs harvested at day 8 and stained for KIND2 and MYH10. Representative fluorescence intensity 3D surface plots of KIND2 signal in non-replated WT-CMs, UNC45B−/−-CMs, and UNC45B−/−:KIND2 OE-CMs. Note that KIND2 and MYH10 IF signals strongly overlap in both WT-CM and UNC45B−/−:KIND2 OE-CMs. (C) Representative IF images of non-replated WT, UNC45B−/−-CMs and UNC45B−/−:KIND2 OE-CMs harvested at day 9 and stained for ACTN2 and F-ACTIN. (D) Rescue efficiency was quantified on the basis of ACTN2 staining. Note that ∼34.7% ± 3.62% of ACTN2 accumulation was rescued in the UNC45B−/−:KIND2 OE-CMs. Results represent data from at least three independent experiments. Scale bars: 10 μm. See also Figure S6.

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  Figure 7. Effect of ectopic UNC45B expression in WT-CMs and UNC45B−/−:-CMs (A) Western blots of the protein expression levels of UNC45B in WT-CMs, WTUNC45B-OE-CMs, and UNC45B−/−:OE-CMs. Exogenous and endogenous UNC45B are indicated. (B) Representative TEM images of WT-CMs and UNC45B−/−:OE-CMs. Scale bars are indicated. (C) Representative IF images of replated WT-CMs and WTUNC45B-OE-CMs harvested at day 30 and stained for the ACTN2 and TNNT2 (upper panel) or for MYH6 and F-ACTIN (bottom panel). Scale bars: 10 μm. Results represent data from at least three independent experiments.